Retroviral infections, including HIV and HTLV, continue to be a pandemic problem. While several drug therapies are able to treat HIV infection, the viral propensity to develop resistance mutations remains challenging. Novel drug targets remain a priority for the HIV treatment arsenal. The most recent class of anti-retroviral drugs target the HIV enzyme integrase. These drugs also inhibit the prototype foamy virus (PFV) integrase. Stable integration of the viral cDNA into the host chromosome is required for a productive retroviral infection and is mediated by an integration complex. The mechanics of monomeric retroviral integrase proteins with model viral and target DNA do not recapitulate the integration reaction of a tetrameric integration complex. Importantly, PFV integrase readily forms physiologically relevant tetramers with model viral DNAs in vitro and is the only integrase where the crystal structure has been solved with viral and target DNAs. We propose to analyze the kinetic and biophysical mechanism of retroviral integration with recombinant PFV integrase as a structural and genetic model. Several innovative single molecule analytical systems will be used to visualize and differentiate the two independent strand transfer events in real time. In addition we will examine for the first time nucleosome DNA targets containing histones with specific post-translational modifications that model chromatin DNA targets in vivo. This proposal contains two highly focused Specific Aims: 1.) Real time analysis of retroviral integration and 2.) Analysis of DNA and chromatin modifications on integration. We will first detail real-time PFV integration into single DNA molecules. Once we have established the kinetics and mechanism of PFV integration into naked DNA, we will introduce increasingly complex physiologically relevant components that include defined poly- nucleosome DNA as well as DNA targets with increased flexibility. Ultimately, this exploratory grant will provide a quantitative and visual platform for probing the detailed kinetics and mechanism of pathogenic retroviral integration, such as HIV.